The conventional technologies to which the present invention pertains will be described below. At present, integrated substrates for bioassay, which are called DNA chips or DNA microarrays (hereinafter generically referred to as “DNA chips”) and on which predetermined DNAs are microarrayed by a microarraying technology, have been utilized for gene mutation analysis, SNPs (monobasic polymorphism) analysis, gene expression frequency analysis, etc., and have come to be used in a wide range of fields including novel medicament development, clinical diagnosis, pharmacogenomics, forensic medicine, etc.
The DNA chip is said to have the characteristic feature that it enables comprehensive analysis of intermolecular reactions such as hybridization since a plurality of kinds of and a multiplicity of DNA oligochains, cDNAs (complementary DNAs), or the like are integrated on a glass substrate or silicon substrate.
An example of the analytical technique using a DNA chip will be described. In the analytical technique, using DNA probes solidified on a glass substrate or silicon substrate, an mRNA extracted from a cell, a tissue, or the like is subjected to PCR amplification while incorporating a fluorescent probe dNTP by a reverse transcription PCR reaction or the like; hybridization is conducted on the substrate; and fluorometry is carried out by use of a predetermined detector.
Here, the DNA chips are classified into two types. A first type is the type in which oligonucleotide is synthesized directly on a predetermined substrate by use of a photolithographic technique applying the semiconductor exposure technique. A representative chip of this type is the chip produced by Affymetrix. This type of DNA chips are high in the degree of integration, but is limited as to the DNA synthesis on the substrate; the length of DNA obtainable is on the order of several tens of bases. A second type of DNA chips is the type also called “Stanford system”, in which the DNA chip is prepared by spotting preliminarily prepared DNAs on a substrate by use of split tip pins and solidifying the DNAs. This type of DNA chips are lower than the first type in the degree of integration, but has the merit that DNA fragments on the order of 1 kb can be solidified.
According to the above-mentioned conventional DNA chip technologies, however, the number of pieces of integration and the integration density of the DNA chip itself have been small. Therefore, the amount of analysis available by one run of assay has not been sufficient. Besides, it has been difficult for the user to freely set the kinds and the number of detection substances and, further, the grouping thereof on the substrate.
In addition, in the conventional DNA chips including the detection substances arrayed on the surface of a substrate having a two-dimensional extension of DNA probes varying (not adjusted) in Tm (melting temperature) or GC content, there has been the problem that there is a high possibility of giving false positive or false negative due to exposure to the same hybridization conditions and washing conditions.
Besides, an assay system for solidifying the detection substances such as DNA probes on the surface of a substrate and for spotting a sample solution containing a target substance and an analyzer also called “reader” or “scanner” for reading the results of the reactions between the detection substances and the labeled target substance have conventionally been fabricated separately and independently. Therefore, it has been impossible to carry out the bioassay process and the subsequent reading and analyzing process in a continuous-form mode. Thus, the conventional system has been very inconvenient to use.
Further, because the amount and shape of the detection substances such as DNA probes and the sample solution droplets have been nonuniform, there has been the technical problem that the accuracy of readout of fluorescence intensity is low.
Moreover, the conventional systems have been high in cost per chip and, further, cost per integration amount, and the analyzer thereof has been very expensive.
Accordingly, it is a principal object of the present invention to provide a bioassay substrate that promises a large integration amount of detection substances solidified and a free grouping of the substances and that is inexpensive, a preferable method of manufacturing the substrate, a bioassay system that makes it possible to perform bioassay efficiently and securely, and a substrate record information readout system that makes it possible to perform an assay process and a reading and analyzing process in a continuous-form mode.